DNA methyltransferases (also referred to as DNA methylases) transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on a DNA molecule. Several biological functions have been attributed to the methylated bases in DNA, such as the protection of the DNA from digestion by restriction enzymes in prokaryotic cells. In eukaryotic cells, DNA methylation is an epigenetic method of altering DNA that influences gene expression, for example during embryogenesis and cellular differentiation. The most common type of DNA methylation in eukaryotic cells is the methylation of cytosine residues that are 5′ neighbors of guanine (“CG” dinucleotides also referred to as “CpGs”). In eukaryotic cells, the methylation of cytosine residues occurs predominantly in CG poor loci (Bird, Nature 321:209, 1986). In contrast, discrete regions of CG dinucleotides called CpG islands typically remain unmethylated in normal cells, except during X-chromosome inactivation and parental specific imprinting (Li, et al., Nature 366:362, 1993) where methylation of 5′ regulatory regions can lead to transcriptional repression (Willson, Trends Genet. 7:107-109, 1991). For example, if a site in the promoter of the gene is methylated, gene silencing is likely to occur.
Improper methylation of DNA is believed to be the cause of some diseases such as Beckwith-Wiedemann syndrome and Prader-Willi syndrome (Henry et a., Nature 351:665, 1991; Nicholls et al., Nature 342:281, 1989). It has also been purposed that improper methylation is a contributing factor in many cancers (Laird and Jaenisch, Hum. Mo. Genet. 3 Spec. No.: 1487-1495, 1994). For example, de novo methylation of the Rb gene has been demonstrated in retinoblastomas (Sakai, et al., Am. J Hum. Genet. 48:880, 1991). In addition, expression of tumor suppressor genes have been shown to be abolished by de novo DNA methylation of a normally unmethylated 5′ CpG island (Issa et al., Nature Genet., 7:536, 1994; Herman et al., Proc. Natl. Acad. Sci., U.S.A., 91:9700, 1994; Merlo et al., Nature Med., 1:686, 1995; Herman et al., Cancer Res., 56:722, 1996; Graff et al., Cancer Res., 55:5195, 1995; Herman et al., Cancer Res., 55:4525, 1995). Many additional effects of methylation are discussed in detail in published International Patent Application PCT/US00/02530.
Current methods used to determine the methylation status of DNA, such as methylation sensitive single nucleotide primer extension (Ms-SNuPE), require a relatively large amount of sample DNA. The amount of DNA in non-invasively collected samples such as blood, urine or shed cells remains the most significant limiting factor in global and targeted methylation studies. In many epigenetic studies, the amount of genomic DNA starting material is limited, especially in experiments utilizing valuable clinical samples, for example oocytes, laser capture microdissected cells, and microscope slides. Current approaches to DNA amplification, such as polymerase chain reaction (PCR) or isothermal whole genome amplification methods, copy the base sequence but do not retain the methylation pattern that was present in the original DNA. Thus, the need exists for methods amplifying DNA while retaining information about the methylation status.